Neural circuits for sexually dimorphic and sexually divergent behaviors in Caenorhabditis elegans

https://doi.org/10.1016/j.conb.2016.02.002Get rights and content

Highlights

  • Several recent advances are driving renewed interest in the C. elegans male.

  • Dimorphic regulation of reproductive muscles facilitates dispersion of progeny.

  • Biological sex ‘tunes’ shared circuits to optimize behavioral strategies.

  • Distributed effects of the sex-determination pathway coordinate sex-typical behavior.

Increasing interest in sex differences in Caenorhabditis elegans neurobiology is resulting from several advances, including the completion of the male tail connectome and the surprising discovery of two ‘new’ neurons in the male head. In this species, sex-specific circuits in the hermaphrodite and male control reproductive behaviors such as egg-laying and copulation, respectively. Studies of these systems are revealing interesting similarities and contrasts, particularly in the mechanisms by which nutritional status influences reproductive behaviors. Other studies have highlighted the importance of sexual modulation of shared neurons and circuits in optimizing behavioral strategies. Together, these findings indicate that C. elegans uses intertwined, distributed sex differences in circuit structure and function to implement sex-specific as well as sexually divergent, shared behaviors.

Introduction

Though the nematode Caenorhabditis elegans has been the subject of extraordinary experimental efforts, the vast majority of this work has focused on just one sex, the hermaphrodite (a self-fertile somatic female). However, increasing interest is being paid to the C. elegans male. A key driver of this is the recent completion of the adult male tail connectome, which has led to important new insights into the neural and genetic basis of sexually dimorphic behavior [1]. Several comprehensive reviews provide overviews of male development and function [2, 3, 4]. Here, we review recent advances in understanding the development and function of sex-specific circuits, as well as work exploring the sexual modulation of anatomically shared circuits.

A well-characterized sex-determination pathway links C. elegans chromosomal sex to the transcription factor TRA-1A, which specifies nearly all sex differences in the soma (see Box 1). In the nervous system, the most obvious aspect of sexual differentiation is in anatomical composition: adult hermaphrodites possess eight sex-specific neurons, while adult males have 91, all of which are integrated into a network of 294 shared neurons [5]. These sex-specific neurons, found largely in the midbody and tail, contribute to sexually dimorphic circuits that subserve male copulatory behavior and hermaphrodite egg-laying. In addition, recent work has highlighted the importance of sex-specific ‘tuning’ of shared neurons and circuits for both sex-specific and sexually modulated behaviors.

The reproductive circuitry of both sexes is optimized to disperse progeny in food-rich environments. When food is scarce, hermaphrodites retain eggs in their uterus [6], and males do not sustain copulation [7]. However, in abundant food, hermaphrodites regulate the frequency of their egg-laying behavior to widely distribute their eggs [8], and males roam to copulate promiscuously [9]. In both sexes, motor regulation is executed by sex-specific muscles derived from the same progenitor cell [10].

The hermaphrodite egg-laying muscles are derived from the embryonic M cell (Figure 1a,b). They consist of 8 uterine muscles, which squeeze the eggs from the uterus, and 8 vulva muscles, which open the vulval slit (Figure 1c). These electrically connected muscles are stimulated by the cholinergic VC and serotonergic HSN neurons, both of which are present only in hermaphrodites (Figure 1d) [11]. The egg-laying circuitry is regulated to allow stochastic dispersion of eggs, presumably to reduce local overcrowding and food competition.

Egg dispersion is accomplished by coupling egg-laying with locomotion. A well-fed hermaphrodite alternates between inactive and active egg-laying states, during which she intermittently lays eggs [8] (Figure 2a). The HSNs modulate the transition between these two states. During the inactive state, the HSNs are down-modulated by the G-protein receptors EGL-47 and EGL-6, the G-protein Go and the hyper-polarizing IRK-1 K+ channel [12, 13]. When the HSNs are active, their secretions synergize with acetylcholine from the VC neurons to adjust the frequency of egg-laying muscle contractions. Genetic analyses indicate that the HSNs’ stochastic activity pattern is regulated by hyperpolarizing chloride conductance through the CLH-3 chloride channels [14]. Analogous to the HSNs, VC secretions are negatively regulated by the small conductance calcium-activated K+ channel KCNL-2; experimentally, channel over-expression causes abnormal egg-laying patterns, mimicking removal of the VCs [15].

VC activity patterns are influenced by locomotion-coupled body bends. Coordinated body wall muscle and uterine/vulval muscle contractions likely force the expulsion of eggs. The VCs are hypothesized to sense postural changes and subsequently secrete acetylcholine, which hypopolarizes the vulva muscles. However, hyperpolarizing current from UNC-103 and other K+ channels maintain the muscles at a sub-firing threshold, keeping them from prematurely contracting. This allows the egg-laying machinery to contract only when the VC-potentiated muscles are co-stimulated by the HSNs (Figure 2a) [16].

Like those in the hermaphrodite, the male sex-specific muscles are derived from the M lineage (Figure 1a,b). However, the male additionally repurposes and incorporates a shared muscle into his reproductive circuitry. In hermaphrodites and larval males, the anal depressor muscle is used for fecal expulsion. However, during development, the sex-determination pathway and signaling from the adjacent M-lineage cells cause the anal depressor to disassemble its dorsal-ventral sarcomere and form a new anterior–posterior sarcomere over the sex muscles (Figure 1b) [17••]. This morphological transformation facilitates intromission [18]. The intromission circuitry shares similar signaling molecules as the egg-laying circuit, but produces different outputs [19, 20]. To intromit his copulatory spicules into his mate, the male uses high frequency protractor muscle contractions to rhythmically thrust his spicules at the vulva. These muscles are excited via electrical connections with the remodeled anal depressor and the gubernacular and oblique muscles, which in turn are stimulated by the glutamatergic and cholinergic PCA, PCB and PCC neurons [1] (Figure 1C,D).

C. elegans hermaphrodites prefer self-fertilization over outcrossing. Recent work has shown that copulation can be physically harmful, damaging the hermaphrodite's cuticle [21], and even causing death [22••, 23••, 24]. N2 hermaphrodites favor self-reproduction and use their chemosensory neurons to sense males and resist copulation [25]. In response, the male persistently thrusts his spicules to breach the reticent hermaphrodite's vulva. The PCA and PCB sensory neurons sense structural features of the vulval lips, and stimulate each other and the spicule associated muscles (Figure 2B). The motor pattern repeats until the spicules penetrate, the male is dislodged, or repeated attempts become futile.

Similar to egg-laying behavior, the male's circuitry uses inhibitory dopamine signaling to dampen motor patterns [26]; however, this occurs when intromission attempts become futile (Figure 2C). The PCA neurons are synapsed onto the glutamatergic chemosensory-interneuron HOA and the dopaminergic ray neurons. Initially, HOA senses the vulva and the dopaminergic ray neurons sense the hermaphrodite cuticle. When penetration attempts initiate, PCA activity is initially moderate; however, its activity increases if attempts are prolonged [18]. At a threshold, PCA neurons are hypothesized to hyper-polarize HOA via the AVR-14 glutamate-gated chloride channel and also stimulate dopamine secretion from the ray neurons. Dopamine then activates the DOP-2 and DOP-3 G-protein receptors on the HOA and PCB neurons. These neurons are electrically coupled via UNC-7 and UNC-9 innexins. Dopamine signaling increases innexin conductance, allowing hyperpolarizing current from HOA to flow into and attenuate PCB. This increases the probability that the male will move off the vulva and search for a more receptive mate [27••].

The circuits that enable sex-specific egg-laying and copulation are interconnected with shared neurons and circuits to allow coordinated behavior. Several studies have identified sexually ‘tuned’ features of these shared components and shown that they are necessary for a variety of sex differences in behavior.

Locomotion is the output of most C. elegans behaviors. However, the relationship between male mating, which requires sustained reverse locomotion (‘response’ behavior), and sex-common motor circuits are poorly understood. Recent work has found that backward locomotion occurs through activation of AVA (a sex-shared reverse command interneuron) by the male-specific PVY/PVX interneurons [28]. These in turn receive direct input from the male-specific tail ray sensilla.

Other shared components of the motor system also have sexually dimorphic properties. For example, ablation of DVA, a sex-shared mechanosensor that regulates body posture [29], has little effect on hermaphrodite locomotion, but causes motor defects in males [30]. This might indicate that DVA inhibits maladaptive sex differences generated by other components of the male nervous system. DVA secretes the oxytocin/vasopressin ortholog nematocin, which promotes sex-specific behaviors [30]; however, DVA's role in locomotion appears independent of this.

The sexes also differ in baseline locomotion: males display greater amplitude of the body wave as well as increased body-bend frequency. These features emerge from complex interactions between body mechanics, neuromuscular physiology, and sex-specific properties of the nervous system [31]. In particular, sex-specific properties of shared sensory neurons control sex differences in body-bend frequency, highlighting the importance of sexual ‘tuning’ of shared sensory function.

Though the male tail is richly endowed with sensory structures, most sensory input is transmitted through sex-shared sensilla in the head. These organs’ anatomy is nearly identical between the sexes. However, multiple studies have identified functional sex differences in sensory function. Sexually dimorphic features of shared neurons, including AWA, AWC, and ASK, have been implicated in olfaction and sex pheromone response [32, 33, 34]. Sex differences in shared circuits are also important for behavioral prioritization. Recent work found that regulation of the food chemoreceptor ODR-10 is important for mediating the choice between feeding and exploration [35••]. In hermaphrodites, high expression of odr-10 in the AWA chemosensory neuron allows efficient food detection. In males, odr-10 expression is low, making animals prone to leave food and search for mates [35••]. Manipulating tra-1 activity (and therefore sexual ‘state’) in this single neuron pair is sufficient to alter the choice between feeding and exploration.

Chemosensory plasticity also differs by sex in C. elegans. NaCl is an attractive cue in both sexes, but hermaphrodites learn to avoid NaCl when it is paired with the aversive stimulus of starvation [36]. Interestingly, this is true for males only if hermaphrodites are absent during conditioning [37]. Otherwise, hermaphrodite cues block starvation-induced NaCl aversion, suggesting that males prioritize the positive association between salt and pheromone. This ‘sexual conditioning’ depends on the male state of the nervous system and may allow animals to efficiently find mates in suboptimal environments [37].

Outside of sex pheromones, little is known about sex-specific communication in C. elegans. Recent work has revealed that the male-specific CEM head sensory neurons secrete extracellular vesicles (ECVs) that contain multiple signaling components [38, 39••]. Interestingly, exposure of males to ECVs promotes copulatory behaviors. The mechanisms by which ECVs are generated and by which they signal remain an area of active study.

Until recently, dogma held that adult males possess 383 neurons, 89 of which are male-specific. Remarkably, in the course of studying a gene important for male exploratory behavior, Sammut et al. [40••] discovered two ‘new’ head neurons, the MCMs (mystery cells of the male). These connect male-specific inputs from the tail to head navigation circuitry and are necessary for NaCl-pheromone sexual conditioning. Surprisingly, the MCMs develop from a male-specific division of the fully differentiated, sex-shared AMso amphid sheath glia during late larval development. This event is controlled cell-autonomously by the sex-determination pathway, as genetic sex-reversal of the AMso glia generates MCMs in hermaphrodites and prevents their development in males [40••].

Aside from the sex-specific deaths of the HSN and CEM neurons [41, 42, 43] and the neurogenesis of the MCMs [40••], relatively little is known about the developmental mechanisms that bring about sex differences in nervous system structure. Recently, Hox-class and TALE-class transcription factors have been shown to be important for male-specific neurogenesis of the CA and CP neurons in the ventral cord [44]. Additionally, VEGF signaling has been shown to interact with plexin and netrin signaling in the migration of male tail sensilla [45]. However, much remains to be learned about the mechanisms that couple tra-1 to the alterations in lineage and cell fate that build sex-specific and sexually ‘tuned’ circuits.

Section snippets

Conclusion

The study of sex differences in neural development and function in C. elegans is uniquely advantaged by the nearly complete knowledge of the cell lineages and connectomes of both sexes. In some cases, downstream effectors of sex determination have been identified, and the mechanisms by which they canalize developmental programs are understood. Studies of the neural logic of sexually dimorphic circuitry has revealed interesting parallels and differences between the sexes, and has shed important

Conflict of interest

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

LRG is funded by the Howard Hughes Medical Institute. DSP is funded by the National Institutes of Health (R01 GM108885) and the National Science Foundation (IOS 1353075).

References (50)

  • J.R. Wolff et al.

    Somatic sexual differentiation in Caenorhabditis elegans

    Curr Top Dev Biol

    (2008)
  • D. Mason et al.

    dmd-3, a doublesex-related gene regulated by tra-1, governs sex-specific morphogenesis in C. elegans

    Development

    (2008)
  • T.A. Jarrell et al.

    The connectome of a decision-making neural network

    Science

    (2012)
  • M.M. Barr et al.

    Male mating behavior

    WormBook

    (2006)
  • S.W. Emmons

    The development of sexual dimorphism: studies of the Caenorhabditis elegans male

    Wiley Interdiscip Rev Dev Biol

    (2014)
  • C. Trent
    (1982)
  • J. Lipton et al.

    Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate

    J Neurosci

    (2004)
  • J.G. White et al.

    The structure of the nervous system of the nematode Caenorhabditis elegans

    Philos Trans R Soc Lond B

    (1986)
  • J.J. Moresco et al.

    Activation of EGL-47, a Gαo-coupled receptor, inhibits function of hermaphrodite-specific motor neurons to regulate Caenorhabditis elegans egg-laying behavior

    J Neurosci

    (2004)
  • L. Emtage et al.

    IRK-1 potassium channels mediate peptidergic inhibition of Caenorhabditis elegans serotonin neurons via a Go signaling pathway

    J Neurosci

    (2012)
  • R. Branicky et al.

    The voltage-gated anion channels encoded by clh-3 regulate egg laying in C. elegans by modulating motor neuron excitability

    J Neurosci

    (2014)
  • C.K. Chotoo et al.

    A small conductance calcium-activated K+ channel in C. elegans, KCNL-2, plays a role in the regulation of the rate of egg-laying

    PLoS ONE

    (2013)
  • K.M. Collins et al.

    Postsynaptic ERG potassium channels limit muscle excitability to alow distinct egg-laying behavior states in Caenorhabditis elegans

    J Neurosci

    (2013)
  • X. Chen et al.

    Developmental alterations of the C. elegans male anal depressor morphology and function require sex-specific cell autonomous and cell non-autonomous interactions

    Dev Biol

    (2015)
  • B. LeBoeuf et al.

    Cell excitability necessary for male mating behavior in Caenorhabditis elegans is coordinated by interactions between Big Current and Ether-A-Go-Go family K+ Channels

    Genetics

    (2012)
  • Cited by (17)

    • One template, two outcomes: How does the sex-shared nervous system generate sex-specific behaviors?

      2021, Current Topics in Developmental Biology
      Citation Excerpt :

      We do not address the contribution of sex-specific neurons to sex-specific behaviors (e.g., HSN and VC motor neurons for egg laying in hermaphrodites), nor the involvement of sex-shared neurons in sex-specific behaviors (e.g. how the shared defecation circuit is integrated into the sperm transfer circuit in males, see Cook et al., 2019; LeBoeuf & García, 2017). Several comprehensive reviews have been written on these topics in recent years (Barr, García, & Portman, 2018; Garcia & Portman, 2016; Oren-Suissa & Hobert, 2017; Portman, 2017). Here we will elaborate on questions regarding common behavioral traits, with special attention to recent advances in the field: Do males and females move differently?

    • Invertebrate Pheromones: Models for Neuroethology

      2019, Encyclopedia of Animal Behavior, Second Edition: Volume 1-5
    • Invertebrate pheromones: Models for neuroethology

      2019, Encyclopedia of Animal Behavior
    • Sexual Dimorphisms: How Sex-Shared Neurons Generate Sex-Specific Behaviors

      2018, Current Biology
      Citation Excerpt :

      First, the species has two sexes: somatically female hermaphrodites that self-fertilize through the transient production of self-sperm, and males that reproduce by mating with hermaphrodites. Second, it exhibits multiple sexually dimorphic behaviors [2,3]. Finally, it has a relatively simple and well-characterized nervous system that can be studied at the single-neuron level.

    • A Single-Neuron Chemosensory Switch Determines the Valence of a Sexually Dimorphic Sensory Behavior

      2018, Current Biology
      Citation Excerpt :

      Sex differences in behavior provide a powerful framework for identifying mechanisms that sculpt naturally occurring behavioral variation [1–4].

    • Sexually Dimorphic unc-6/Netrin Expression Controls Sex-Specific Maintenance of Synaptic Connectivity

      2018, Current Biology
      Citation Excerpt :

      Much less is known about dimorphic anatomical or molecular features of neurons that are present in both sexes. The nervous system of C. elegans offers the unprecedented opportunity to study sexual dimorphisms in nervous system anatomy with the resolution of single cells and synapses [5–7]. Electron micrographical reconstructions of the connectome of the C. elegans male and hermaphrodite (a somatic female) revealed that some sex-shared neurons are wired to one another in a sexually dimorphic manner [1, 2] (Figure 1A).

    View all citing articles on Scopus
    View full text